JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p. 608-612 Vol. 32, No. 3 0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology Detection of Microsporidia by Indirect Immunofluorescence Antibody Test Using Polyclonal and Monoclonal Antibodies ABDALLA M. ALDRAS,' JAN M. ORENSTEIN,2 DONALD P. KOTLER,3 JOHN A. SHADDUCK,4 AND ELIZABETH S. DIDIER1* Department of Microbiology, Tulane University Regional Primate Research Center, Covington, Louisiana 704331; Department of Pathology, George Washington University Medical Center, Washington, D.C. 200372; St. Luke's/Roosevelt University Medical Center, New York, New York 100253; and Department of Veterinary Pathobiology, Texas A & M University, College Station, Texas 778434 Downloaded from Received 5 August 1993/Returned for modification 4 October 1993/Accepted 17 November 1993

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and Ell, and were found to secrete MAbs of the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By

Western blot immunodetection, the three MAbs detected three E. helkm antigens with relative molecular http://jcm.asm.org/ weights (Mrs) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with Mrs of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. helem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. heUlem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that

polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. helkm, can be used for on September 12, 2018 by guest indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.

Microsporidia are small obligate intracellular protozoan AIDS patients whose immune responses are compromised (12, parasites which infect a wide range of animal hosts, including 13). Mammalian microsporidium spores do stain with Gram, all classes of vertebrates and most invertebrates (8). Awareness Giemsa, calcofluor, and concentrated trichrome (25, 30, 32, 33, of microsporidia is important because the number of reported 35), but because these organisms are very small (measuring 0.5 microsporidiosis cases in AIDS patients is increasing (2, 5-7, to 2.0 ,um by 1.0 to 4.0 ,um) they are difficult to distinguish from 22, 23, 29). The three most common microsporidia reported to bacteria and small yeasts. In this article, we report the use of infect individuals with AIDS are Enterocytozoon bieneusi, En- polyclonal antibodies and monoclonal antibodies (MAbs) in cephalitozoon hellem, and the Encephalitozoon-like Septata detecting microsporidia in formalin-fixed stool and urine by intestinalis. E. bieneusi primarily infects small-intestinal entero- the indirect immunofluorescence antibody test (IFAT). cytes, causing diarrhea, but can also infect the biliary tract, leading to cholangitis (4, 10, 19, 23, 26). On the other hand, E. hellem and the Encephalitozoon-like S. intestinalis are not as MATERIALS AND METHODS tissue specific and have been reported to cause keratoconjunc- Parasites. E. corneum were tivitis, sinusitis, nephritis, and enteritis (3, 15, 22, 27). Although hellem, E. cuniculi, and Nosema grown in Madin-Darby canine kidney (MDCK) cells by using cases ofEncephalitozoon cuniculi-associated hepatitis and peri- tonitis in RPMI 1640 supplemented with 5% fetal bovine serum, strep- AIDS patients have been reported, it is possible that tomycin (100 ,ug/ml), penicillin (100 U/ml), amphotericin B they were actually due to the morphologically identical E. hellem. (0.25 ,ug/ml), and L-glutamine (2 mM) (referred to as complete as described Parasites from Presently, definitive identification of microsporidiosis de- RPMI) previously (11, 14). tissue culture supernatants were centrifuged at 400 x g for 15 min, pends upon transmission electron microscopy, which is time- and In washed once in 25 mM Tris-buffered saline (TBS; pH 7.4) cost-consuming. addition, transmission electron micros- containing 0.05% Tween 20 (TBS-Tween), resuspended in copy may not be sensitive enough to detect small numbers of organisms. Serological studies for detecting microsporidium- TBS, and centrifuged over 50% Percoll (Pharmacia, Piscat- away, N.J.) at 14,000 x g for 2 min. The parasites were washed specific antibodies are reliable for antemortem diagnosis in twice with TBS to remove the Percoll and stored at 4°C in infected laboratory animals (29, 30) but may be unreliable for bicarbonate buffer (pH 9.6) for use in the enzyme-linked immunosorbent assay (ELISA) or were stored in TBS for all other uses. * Corresponding author. Antibodies. Murine hyperimmune antisera were obtained

608 VOL. 32, 1994 DETECTION OF MICROSPORIDIA BY IFAT 609 from female BALB/c mice (Charles River Laboratories, Wilm- natant. After overnight incubation the strips were washed ington, Mass.) immunized intraperitoneally with four inocula- three times with TBS-Tween and incubated with alkaline tions of 5 x 107 spores of E. hellem, E. clinicutli, or N. corneum phosphatase-conjugated goat anti-mouse IgG-lgM (Sigma) at 4-week intervals. Sera were collected by tail bleeding 1 week diluted 1:1,000 in TBS-BSA. After incubation for 60 min at after each inoculation, pooled from mice within each group, 37°C, the strips were washed with three changes of TBS and and stored at - 70°C until used. Sera from the third bleeding rinsed with developing buffer (50 mM Tris-HCl [pH 9.6] were used in these studies, and the ELISA titers obtained by containing 3 mM MgCl,) as described previously (1). Bands assaying the sera against each homologous microsporidian were visualized by the addition of the substrates 5-bromo-4- were -1: 12,800. chloro-3-indolylphosphate (0.05 mg/ml) and nitroblue tetrazo- The E. liellem-immunized BALB/c mice used for obtaining lium (0.01 mg/ml) to the developing buffer. After color devel- the polyclonal antisera were then employed for producing opment for 30 min, the strips were rinsed in distilled water, MAbs. Four days after the fourth inoculation, spleen cells were dried, and stored in the dark. fused with the murine myeloma cell line X63.Ag8.653 (17). IFAT. The IFAT was performed as described previously Approximately 2 weeks later, and after at least three medium (17). Thin smears were prepared on microscope slides from (i) Downloaded from changes, the supernatants were screened for antibody produc- patient stools containing E. bieneusi fixed with buffered forma- tion by ELISA by using intact E. hellem spores as antigens. lin-stool (1:3, vol/vol); (ii) urine specimens containing S. Positive hybridomas were cloned twice by limiting dilutions intestinalis, which were centrifuged at 400 x g for 10 min and and seeded at 105 cells per ml of complete RPMI (containing resuspended in 1/50 of the original volume of buffered forma- 10% fetal bovine serum) into 25-cm2 tissue culture flasks. lin; (iii) formalin-fixed tissue culture-derived E. hellem, E. Three-day log-phase culture supernatants were used in the ciuniculi, or N. corneum; and (iv) nonfixed microsporidia assays described here. The MAb isotypes were determined derived from tissue culture. The slides were air dried, fixed in with the Sigma immunotype kit by following the directions 100% methanol for 5 min, and then incubated with MAbs, enclosed (catalog no. ISO-1; Sigma, St. Louis, Mo.). polyclonal antisera, or a negative hybridoma supernatant. Ascites fluid was produced in BALB/c mice by intraperito- After incubation in a moist chamber for 30 min at 37°C, the http://jcm.asm.org/ neal injection of 0.5 ml of pristane (2,6,10,14-tetramethylde- slides were washed twice in TBS. Fluorescein isothiocyanate- canoic acid) followed by intraperitoneal injection of 10" hybri- conjugated goat anti-mouse IgG-IgM-IgA (Sigma) was ab- doma cells 2 weeks later (17). The ascites fluid was tapped sorbed with formalin-fixed stool sediment, diluted 1:200, and approximately 3 weeks later, centrifuged (400 x g for 15 min) added to the slides for incubation at 37°C for 30 min. Then the to remove cells, aliquoted, and stored at - 70°C until used. slides were washed, coverslips were added with Cytoseal ELISA. To perform the ELISA, E. hellem, E. cuniculi, and mounting medium (Stephens Scientific, Riverdale, N.J.), and N. corneum in 0.2 M bicarbonate buffer (pH 9.6) were used to the slides were examined under UV light microscopy (Olym- coat 96-well, flat-bottom polystyrene microtiter plates (Corn- pus AH2 microscope) with an excitation wavelength of 380 to ing Glass Works, Corning, N.Y.) at a concentration of 5 x 107 490 nm, which generates an observation light wavelength equal spores per well as described by Hollister and Canning (18). to or greater than 515 nm. on September 12, 2018 by guest MDCK cell proteins were adjusted to 1.0 mg/ml in bicarbonate buffer. Plates were incubated overnight at 37°C, washed with RESULTS TBS three times, and blocked with 3% (wt/vol) bovine serum albumin (BSA) in TBS for 2 h at 37°C. After three washings Characterization of MAbs by ELISA. Three hybridoma with TBS, 100 RI of the appropriate undiluted hybridoma or cultures, whose supernatants reacted most strongly to E. control supernatant was added to each well. The plates were hellem in the ELISA, were cloned twice, designated C12, E9, incubated for 2 h at 37°C, washed three times in TBS-Tween, and El1, and found to secrete MAbs of the IgM isotype. The and incubated for 1 h at 37°C with alkaline phosphatase- MAbs were then tested again by ELISA using E. hellem as well conjugated goat anti-mouse immunoglobulin G (IgG)-IgM- as E. cuniculi and N. corneum as antigens (Table 1). The three IgA (Sigma) diluted 1:4,000 in TBS containing 1% (wt/vol) MAbs reacted most strongly against E. hellem. C12 and E9 also BSA (TBS-BSA). After the plates were washed three times reacted strongly to E. cuniculli and less strongly against N. with TBS, 100 p.1 of the enzyme substrate p-nitrophenylphos- corneum, a microsporidian isolated from the corneal stroma of phate (1.0 mg/ml of diethanolamine buffer, pH 9.6) was added, a human immunodeficiency virus-seronegative individual (9, and the optical densities were read at 405 nm on an ELISA 31). ElI produced nearly identical low levels of reactivity spectrophotometer (Dynatek Laboratories Inc., Chantilly, Va.) against both E. cuniculi and N. corneum. In all cases, however, (17). the MAbs produced at least threefold-higher levels of reactiv- SDS-PAGE and Western blotting (immunoblotting). Para- ity against the microsporidia than against MDCK tissue culture site proteins were separated by sodium dodecyl sulfate-poly- host cell proteins or against a negative hybridoma control acrylamide gel electrophoresis (SDS-PAGE) by the method of supernatant. Laemmli (20), using a 5% stacking gel, a 10 to 20% gradient Characterization of MAbs by Western blot immunoassay. resolving gel, and diallytartardiamide as the cross-linker. Par- Western blot immunodetection of E. hellem was performed to asites in sample buffer containing r3-mercaptoethanol were determine the molecular weights of proteins recognized by the boiled for 5 min and centrifuged at 14,000 x g for 1 min to MAbs. All three IgM MAbs detected proteins with MrS of 62, remove particulate materials. Each preparative slab gel (16 by 60, and 50 at the highest dilutions producing reactivity. Less 20 cm) was loaded with 2 x 109 parasites. After electrophore- diluted hybridoma supernatants also bound to proteins with sis, the separated polypeptides were electrophoretically trans- Mrs of 55 and 53 (Fig. 1). Identical results were obtained when ferred onto Immobilon-P membranes (Millipore, Bedford, parasite proteins were prepared by using the protease inhibi- Mass.) and cut into strips. The strips were incubated with 5% tors leupeptin (0.5 ,ug/ml) and phenylmethylsulfonyl fluoride (wt/vol) nonfat dry milk (Carnation) in TBS for 30 min to block (0.2 mM). unbound sites, washed in TBS-Tween for 10 min, and incu- IFAT. Murine polyclonal antisera to E. cuniculi and E. bated with various dilutions of each hybridoma supernatant, hellem, as well as the MAbs raised against E. hellem, were used positive control serum, or negative control hybridoma super- to detect microsporidia by IFAT. Smears of tissue culture- 610 ALDRAS ET AL. J. CLIN. MICROBIOL.

TABLE 1. ELISA reactivities of MAbs raised to E. hellema ELISA values for absorbance to antigen target" MAb E. hellem E. cuniculi N. corneum MDCK cells C12 1.153 ± 0.011 0.785 ± 0.090 0.555 ± 0.005 0.101 ± 0.006 E9 0.953 ± 0.058 0.491 ± 0.014 0.341 ± 0.070 0.094 ± 0.002 Eli 0.818 ± 0.072 0.310 ± 0.033 0.318 ± 0.043 0.052 ± 0.013 Neg. sup't. 0.104 ± 0.007 0.055 ± 0.030 0.020 ± 0.027 -0.045 ± 0.0001 a Undiluted culture supernatants from twice-cloned hybridomas were recovered and assayed as described in Materials and Methods. b Values are the means of two replicates ± the standard deviations.

derived E. hellem, E. cuniculi, N. corneum, formalin-fixed hellem (diluted 1:500) detected both native E. hellem (data not Downloaded from stools mixed with the tissue culture-derived organisms, and shown) and formalin-fixed E. hellem (Fig. 2A) and E. cuniculi stool and urine specimens with E. bieneusi and S. intestinalis, (data not shown), as well as E. bieneusi (Fig. 213) and S. respectively, were examined. The murine antiserum to E. intestinalis (Fig. 2C) in patient stools and urine, respectively. The murine polyclonal antiserum to E. cuniculi produced results identical to those obtained with the murine polyclonal antiserum to E. hellem (data not shown). The three MAbs E producing the strongest reactivity to E. hellem in the ELISA to . _ also detected E. hellem, E. cuniculi, E. bieneusi, and S. intesti- ci :} nalis but not N. corneum by IFAT. Results are shown for E9 http://jcm.asm.org/ C,' a) dL detection of E. hellem (Fig. 2D), E. bieneusi (Fig. 2E), and S. 0 C.) iJ Vii UJ E kDa intestinalis (Fig. 2F). IFA staining ofE. bieneusi with the MAbs (Fig. 2E) was not as strong as with the polyclonal antisera (Fig. 2B). The MAbs did not stain Cryptosporidium, Isospora, Giar- dia, Candida, or Trichomonas spp. in known positive patient stool specimens (data not shown). The murine hyperimmune sera raised against E. hellem or E. cuniculi did detect N. corneum and Candida albicans when used at dilutions of 1:200 or lower, and the nonspecific binding to yeast cells could be reduced by absorbing with C. albicans (data not shown). In addition, background staining, particularly when the polyclonal on September 12, 2018 by guest _ 106 antisera were used, could be further reduced by absorbing the - 80

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FIG. 1. Western blot characterization of MAbs to E. hellem. E. hellem proteins were separated by SDS-PAGE under reducing condi- tions and transferred to Immobilon-P membranes. The membrane antigen strips were incubated with MAbs (C12, E9, and Eli) harvested from 3-day log-phase hybridoma cell cultures and diluted 1:10; this was followed by immunodetection using alkaline phosphatase-conjugated goat anti-mouse IgM and color development with nitroblue tetrazo- lium and 5-bromo-4-chloro-3-indolylphosphate. Ascites fluid from FIG. 2. IFAT for detecting microsporidia using polyclonal antibod- BALB/c mice inoculated with hybridoma E9 cells was diluted 1:100, ies and MAbs. Formalin-fixed tissue culture-derived E. hellem (A and and the hyperimmune murine polyclonal antiserum to E. hellem (m a D), E. bieneusi in stool (B and E), and S. intestinalis in urine (C and F) E. hellem) was used at a dilution of 1:2,000. Molecular masses of were stained with murine hyperimmune polyclonal antiserum raised standards are given at the right (in kilodaltons). Neg. sup't., negative against E. hellem (A to C) or MAb E9 (D to F) as described in supematant. Materials and Methods. VOL. 32, 1994 DETECTION OF MICROSPORIDIA BY IFAT 611 fluorescein isothiocyanate-conjugated antibodies with forma- we found that the polyclonal antisera generated more back- lin-fixed stool sediment. ground in formalin-fixed stool specimens with the IFA stain. The degree of background depended on the dilution of anti- DISCUSSION serum, as also described by Weiss et al. (36). We also found that the polyclonal antisera stained yeast cells, but this problem Opportunistic infections with microsporidia in AIDS pa- could be overcome by absorbing the antisera with C. albicans. tients are increasingly being reported, yet the number of Neither the Candida-absorbed polyclonal antisera nor the microsporidiosis cases is probably greatly underreported. Mi- MAbs stained yeast cells, bacteria, or Cryptosporidium, Iso- crosporidium infections are difficult to diagnose, primarily spora, Giardia, or Trichomonas spp.; both the antisera and because the organisms are small and difficult to distinguish MAbs thus appeared to be specific for microsporidia. In from bacteria and small yeasts in tissue and stool. Giemsa stain addition, we found that absorbing the fluorescein isothiocya- (25, 32) and a modified trichrome stain using chromotrope 2R nate-conjugated antibody with sedimented stool material (35) have been used to detect microsporidia in stool, but with helped reduce the background staining. some difficulties. Giemsa-stained microsporidia are blue and That the most strongly reacting MAbs selected for cloning Downloaded from display a purple-blue nucleus which distinguishes them from were of the IgM isotype was probably due to the pentameric bacteria. It is difficult, however, to find microsporidia in stool structure of the IgM antibodies, which could amplify the signal smears in which most other organisms also stain blue. The in the ELISA more than monomeric IgG MAbs could. Al- modified trichrome (chromotrope 2R) staining method de- though the three MAbs appeared to detect the same proteins scribed by Weber et al. (35) has the advantage that most on Western blots of E. hellem, binding by E9 and E1i (but not bacteria counterstain light green, leaving the microsporidia C12) was lost by periodate oxidation, suggesting that the pink. The stool smear must be very thin in order to observe the epitopes recognized by E9 and El1 are carbohydrates (unpub- internal structure of the microsporidia, so microsporidia may lished observations). In addition, the ELISA values for absor- be missed if the parasite burden is low or if microsporidia are bance by the MAbs against the different microsporidia varied, mixed with mucus. In addition, small yeasts and some bacteria so that it is possible that the MAbs detected different epitopes http://jcm.asm.org/ in stool also stain pink, which can complicate the results. on the same proteins. The fact that several proteins were Finally, it is crucial to monitor the destaining step so that the detected by each MAb could be due to the presence of the microsporidia remain pink but high background levels of same epitopes on precursor molecules and/or breakdown staining are not generated (16). A calcofluor staining method products in the parasite preparations used to prepare the blots. utilizing Uvetix 2B (Ciba-Geigy) or fluorescent brightener Adding the protease inhibitors leupeptin and phenylmethylsul- (catalog no. F-6259; Sigma) also may be useful for detecting fonyl fluoride to the parasite preparations, however, still microsporidia (33). The microsporidia display a relatively thick resulted in detection of the multiple bands. Finally, it is ring of fluorescence, and the anterior region appears concave. possible that the same epitope is found on functionally differ- However, because yeasts also stain with calcofluor, further ent proteins. studies are needed to assess the specificity and reliability of The availability of polyclonal antibodies to Encephalitozoon on September 12, 2018 by guest these stains. species and MAbs to E. hellem should be particularly useful in The use of microsporidium-specific antibodies in IFAT screening specimens for the presence of the most common procedures appears to overcome some of these difficulties. In microsporidia causing opportunistic infections in AIDS pa- earlier studies, IFAT procedures with polyclonal antisera were tients. Additional specimens need to be stained with these used to show that several species of microsporidia demon- reagents to determine their feasibility and reliability for rou- strated immunological cross-reactivity (21). More recently, tine diagnostic and epidemiologic studies. In addition, IgG polyclonal antisera produced against E. cuniculi and E. hellem MAbs which, in combination, may prove more useful than IgM were used to diagnose ocular and systemic E. hellem infections MAbs are being evaluated. Most of the MAbs generated (27, 28), and polyclonal antiserum raised against E. cuniculi in against E. hellem were of the IgM isotype, and these MAbs rabbits was used in the IFAT to detect E. bieneusi organisms in reacted more intensely than the IgG MAbs. However, we are deparaffinized tissue sections (36) and in stool (37). finding that the use of the IgM MAbs characterized in this In this study, we observed that polyclonal antisera raised study may not be feasible for ELISA procedures because of against E. cuniculi and E. hellem in mice, as well as the three high-level, nonspecific reactivity. This may be due to the higher MAbs, detected formalin-fixed, tissue culture-derived Enceph- level of sensitivity in ELISA than in IFA staining. Another alitozoon species as well as E. bieneusi in formalin-fixed stool. difficulty is that the secondary or conjugated antibodies need to The polyclonal antisera to E. cuniculi and E. hellem also be absorbed with sedimented stool to reduce background in detected a newly described microsporidian, S. intestinalis (3, the IFAT, and it may not be possible to remove this interfer- 24), in formalin-fixed urine. The antiserum raised against E. ence for the ELISA. To reduce this background interference, cuniculi and E. hellem detected N. corneum only when used at one could conjugate the MAb (e.g., with fluorescein isothio- lower dilutions, suggesting that E. hellem and E. cuniculi are cyanate or alkaline phosphatase), which is easier with IgG than more closely related to E. bieneusi and S. intestinalis than to N. with 1gM MAbs. The polyclonal antibodies and MAbs de- corneum. Unlike the polyclonal antisera raised against E. scribed in this study, however, provide a basis for screening cuniculi and E. hellem, the hybridoma supernatants did not specimens for the presence of microsporidia by IFA staining. stain N. corneum or yeast cells in the IFAT, even though they Species-specific diagnosis of microsporidium-positive speci- did react somewhat to N. corneum in the ELISA. Microspo- mens should then become possible as species-specific immu- ridia of the genus Nosema typically infect insects, and the lack nologic and molecular probes become available. of cross-reactivity in the IFAT is most likely due to a lower degree of phylogenetic relatedness (34). ACKNOWLEDGMENTS The MAbs and polyclonal antibodies provided different We thank Donna Bertucci for excellent technical assistance and advantages in the IFA stain. E. bieneusi stained more intensely Murphy Dowous for photographic assistance. with the polyclonal antisera raised against E. cuniculi or E. This study was supported by grants EY08778 and RR00164 from the hellem than with the MAbs raised against E. hellem. However, National Institutes of Health. 612 ALDRAS ET AL. J. CLIN. MICROBIOL.

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